During abstinence from alcohol, stress in alcoholics can result in negative affect and craving (1), a response which is accompanied by a change in brain functional magnetic resonance imaging (fMRI)-effects of stress not seen in social drinkers (2). To model the negative affect induced by stress during abstinence in alcoholics, an extended period of chronic intermittent alcohol (CIA) exposure was found to cause an enduring adaptation that sensitizes stress-induced negative affect during abstinence. These series of clinical and basic findings are consistent with the kindling/stress hypothesis of alcoholism. Even though the central amygdala (CeA) is known to play an important role in stress-induced negative affect after CIA exposure, unknown is the role input circuits to the CeA have in facilitating this stress-induced anxiety. In addition to CRF, other inputs that synapse on CeA neurons are glutamate (GLU) terminals from the basolateral amygdala (BLA), oxytocin (OXY) terminals from the hypothalamus, and vasopressin (VP ) terminals. Nonetheless, the influence these other inputs have on stress-induced anxiety after CIA exposure has not been identified. Likewise unidentified is whether CeA medial division (CEM) neurons that output terminals to the periaqueductal gray (PAG) and other sites are a critical component of the neural circuit that supports stress-induced anxiety associated with CIA. To guide addressing these unknowns, the hypothesis tested is that stress facilitates anxiety during abstinence from CIA exposure by influencing a neural circuit composed of terminal inputs to the lateral (CEL) and medial (CEM) divisions of the CeA that modulate CEM output. First, to permit optogenetic investigations of the potential role BLA terminals have in facilitating stress-induced anxiety after CIA exposure, an AAV5-eYFP vector with a CAMKII promoter containing the rhodopsin derivatives halorhodopsin (NpHR3.0) or channel- rhodopsin (ChR2) will be placed into the BLA to confirm localization of GLU terminals from the BLA to the lateral (CEL) and the medial (CEM) divisions of the CeA. Subsequently tested with optogenetics will be whether ChR2 activation or NpHR3.0 inhibition of BLA terminal release of GLU on CEL neurons will influence facilitation of stress-induced anxiety after CIA exposure. This latter optogenetic strategy will be complemented by determining if activation of CEL neurons with an OXY receptor agonist will block facilitation of stress-induced anxiety after CIA exposure. Upon completing investigations of CEL neural inputs, optogenetic inhibition or excitation of GLU-containing BLA terminals synapsing on CEM neurons will assess if the stress-induced anxiogenic action after CIA exposure is affected. Because VP-containing terminals synapse on CEM neurons to induce anxiety- like behavior, it is reasoned that activation of CEM neurons with VP may contribute to the anxiety induced by stress following CIA exposure. To confirm a direct involvement of CEM neurons in VP action, an AAV-eYFP vector with the NpHR3 expressed in CEM neurons will test if optogenetic inhibition of CEM neurons will alter both stress- as well as VP-induced anxiety after CIA exposure. To confirm that VP action contributes to stress-induced anxiety after CIA exposure, VP receptor subtypes will be pharmacologically antagonized in the CEM prior to stress to determine if the stress- induced anxiety after CIA exposure is prevented. Upon confirmation of CEM terminal presence in the PAG, involvement of these CEM terminals in the PAG in stress- and VP-induced anxiety observed after CIA exposure will be assessed by optogenetic inhibition or excitation of these CEM terminals- an approach to implicate both the CEM and the PAG in facilitation of the stress-induced anxiety after CIA exposure. Collectively, the proposed pharmacological and optogenetic strategies utilized can be expected to define whether involvement of specific neural inputs to the CEL and CEM, which are accompanied by CEM output to the PAG and other sites, form a circuit that influences facilitation of stress-induced negative affect after CIA exposure. This proposed circuit involving the CeA is felt to be associated with the neuropathology responsible for the facilitated negative affect observed to stress that facilitates craving in alcoholics durin abstinence. New knowledge concerning adaptations in this circuit after CIA exposure could possibly provide clues by which to minimize the barrier to developing new and improved therapeutics to treat the negative symptoms to stress observed in abstinent alcoholics.